The long term objective of this grant is improved understanding of how alcohol perturbs fetal folate nutrition. Data generated in the course of the Principle Investigators research has included biochemical characterization of folate binding proteins (FBPs) isolated from human placenta and milk. Subsequent studies have identified that membrane-associated FBPs are specific transport proteins responsible for folate uptake in intact malignant and normal human cells. It is therefore appropriate to extend such studies to models (with partial or complete organs) that simulate the in vivo situation. Such a model is offered in the in vitro human single placental cotyledon perfusion system with which the Co-investigator has extensive practical experience. Preliminary collaborative efforts have successfully demonstrated that this model can be used to analyze transplacental folate transport, which alcohol acutely perturbs. The hypothesis that placental folate receptors (PFRs) are functionally active in transplacental folate transport will be tested after definition of the kinetics of transport (placental uptake and fetal folate delivery) with respect to extracellular folate concentration dependence, as well as studies that evaluate the influence of folate receptor occupancy by ligand. Biochemical methods to specifically perturb PFRs at their folate binding sites both competitively (using various folate analogues and specific anti-PFR antibodies) and non competitively (using an N-hydroxysucimimide ester of folate which specifically and covalently interacts with PFRs) will be carried out. An additional mechanism, that of passive diffusion of folates across the placenta at high extracellular folate concentrations (which has precedence in isolated human cells) will also be investigated after PFRs are inactivated. Such alcohol acutely increases fetal folate delivery, studies will be directed at determining if this is due to an effect on PFR-mediated or passive diffusion mechanisms. Additional biochemical studies on cultured placental cytotrophoblasts using biosynthetic methods will determine whether the origin of soluble FBPs is from membrane associated PFRs. Finally, the membrane-associated PFRs (hydrophobic FBPs) as well as the placental protease that cleaves hydrophobic FBPs to soluble (hydrophilic) FBPs will be isolated and characterized. Such studies will lay the foundation for further investigation on the chronic effects of alcohol on ultimate fetal folate delivery on primates and placentas of pregnant alcoholics.